This is my enzyme assay averages. According to the numbers the values are linear, whcih shows that the assay worked.
This is my FPLC result. Unfortunately I lost my protein here. There was supposed to be a peak at lanes 34-36 but there weren't any peaks. The only peaks were the result of residue from previous runs.
WEEK 12
This is my dried page gel. Pretty much the same thing as my gel when it was soaking in water except now it is now a bit more distinct. Elution 5, which contains my protein, is barely visible compared to the other bands. Because of this I had to concentrate my protein to 1ml from 5ml.
My SDS page gell. Starting from the left, Lane 1 is the protein marker, lane 2 was skipped, lane 3 is sample 2, lane 4 is sample 3 (flow through), lane 5 is sample 4 (wash), lane 6 is sample 5 (elution 1) and lane 7 is sample 6 (elution 2) but nothing showed up on that lane.
This is my nanodrop of elution 1. As it can be seen my concentration of protein is quite low, which is why there is a very thin band in my SDS page gell where the elution one band rests.
WEEK 11
This week I finished my PCR squared and completed my PCR cleanup
And I finished up my protein expression.
The Pellet Weight was 2.07g for sample 1 and 2.54 for sample 2.
Absorbance reading to get to 0.1 at 600nm. This was done before trying to get to 0.3.
It took an hour and thirty minutes to get to get to 0.1 absorption
Sample 1 Sample 2
3:00
0.163 0.108
0.193 0.199
0.29 0.28
This is the absorption of 0.3 at 600nm.
I forgot to take a picture of the pellet but I completed the procedure and the supernatant is in the 4 degree fridge. Also I had two samples but ended up adding too much lysate to sample one so I did my protein purification with sample 2.
WEEK 10
I was not in lab at all this week, well maybe for like 2 to 3 hours, but i will make the hours up for next week. Had three tests and I got pretty sick so sorry about that.
I was supposed to do PCR squared but I will be finishing that next week.
I did my slides for my presentation and talked to Dr.B about them.
Finally fixed my virtual screening.
I started on my Protein Expression but I left too long of a gap so I need to redo it for next week.
So for next week I will finish Virtual Screening of HF9 Library and most probably one other library now that I can do it myself.
Present
Finish all of the PCR
Finish Protein Expression
Will definitely start cloning but don't know if I will be able to finish.
WEEK 9
Mohammed - I think this should be Week 9 - spit up your Week 8 and 9 results.
Good job on the cloning! - Dr. B
This is the 1st gel run with the first and second PCR runs. The first PCR showed up succesfully as it smeared but unfortunately the second one showed one band but it wasn't very distinctive. This may be due to the fact the blue juice used (6x blue juice) may not have been the right blue juice to use.
Lane 4: 100bp ladder
Lane 5: 2nd PCR
Lane 6: 1st PCR
Lane 7: 1000kb ladder
Lane 8: 1st PCR
This was the second gel for the 1st and 2nd PCR. Again the first PCR showed up but unfortunately the 2nd PCR didn't show up again even though a different blue juice was used. So I have no idea what is happening.
Lane 1: 100 bp ladder
Lane 2: 1000 kb ladder
Lane 3: Primary PCR
Lane 4: Secondary PCR
IT WORKEDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD
Lane 1: 100bp ladder
Lane 2: 1kb ladder
Lane 3: 1st PCR
Lane 4: 2nd PCR
WEEK 8
Also ran the first GOLD job. Here is the gold.conf file for the first run.
GOLD CONFIGURATION FILE
AUTOMATIC SETTINGS
autoscale = 0.1
POPULATION
popsiz = auto
select_pressure = auto
n_islands = auto
maxops = auto
niche_siz = auto
GENETIC OPERATORS
pt_crosswt = auto
allele_mutatewt = auto
migratewt = auto
WEEK 7
Did the Primer Overlap Assembly as I designed a set of forward and reverse primers for PCR for serine threonine phosphotase 2C
This is the DNA sequence for the particular target.
This is the oligonucleotides(primers) that I obtained
The protein is serine throenine phosphotase 2C and the strain was made for E.Coli 21
For the particular target there are 24 primers
Also in wet lab I completed the primary and secondary PCR and will do PCR squared this coming week.
WEEK 2
This was the first PCR. As it can be seen only the ladder showed up, which means the gel was made correctly but something else went wrong. It may have been incorrect pipetting techniques or other human errors.
WEEK 2 (PCR WITH pGBR22) Lane 1: skip Lane 2: 100 bp ladder Lane 3: PCR of pGBR22 Tube A Lane 4: PCR of pGBR22 Tube B Lane 5: PCR of pGBR22 Tube C Lane 6: PCR of pGBR22 Tube D
WEEK 2 (ANALYZING DNA SEQUENCES)
HUMAN SEQUENCING
NUCLEOTIDE SEQUENCING
COMPARISON OF PURPLE PROTEIN TO pGBR22 a
WEEK 2 RE DIGEST
Lane 2 1kB ladder
Lane 3 uncut pgbr22 plasmid
Lane 4 eco R1
Lane 5 PvuII
Lane 6 EcoR1 + PvuII
WEEK 3 SUBMITTING DNA SEQUENCE
The Requests are entered, your Order Number is: 72169
University of Texas ICMB Core Facilities DNA Sequencing 2500 Speedway MBB 1.426L, A4800 Austin, TX 78712 512 475-7844 begin_of_the_skype_highlighting512 475-7844end_of_the_skype_highlighting p 512 471-2149 fax
Grant Account Number || ||
26-6815-3050
The order contains 2 Samples.
If submitting 96 well plates, please write ORDER NUMBER on the plate.
Sample
Conc
DNA Type
Size
Primer
Primer Conc.
1
MA1
27
pGBR22
4000
M13For-20
5
2
Ma2
27
ppGBR22
4000
M13Rev-24
5
WEEK 3 (PCR WITH pGBR22 AGAIN)
_
This is the second gel for PCR with pGBR22. Lane 6 vanished completely and this may have been due to incorrect pipetting techniques as the gel may have been pierced at the bottom. Also lanes 4 and 5 look exactly similar which means that the sample from either lane 5 went to lane 6 or vice versa. Lane 3 is very blurry as it did not run correctlyl.
Lane 2 1kB ladder
Lane 3 sample a
Lane 4 sample b
Lane 5 sample c
Lane 6 sample d
WEEK 3 (PCR WITH PNIC BSA4)
Although the bands didn't show (except for lane 4) it wouldn't have mattered as the same concentration was used instead of different ones.
Lane 2: Ladder 100bp
Lane 3: 1 in 1000 concentration
Lane 4: 1 in 1000 concentration
Lane 5: 1 in 1000 concentration
WEEK 4 (PCR WITH PNIC BSA4)
_
The gen lanes had actually messed up but it was only the last two lanes. Lanes 2-6 did not show at all. This may have been due to incorrect pipetting techniques of the gel may have been more damaged than it was thought.
Lane 1: 100bp ladder Lane 2: 1 in 100 concentration Lane 3: 1 in 1000 concentration Lane 4: 1 in 10000 concentration
ALSO IN WEEK 4 COMPLETED THE PYMOL REFRESHER WHICH IS IN GOOGLE DOCS
WEEK 5(PCR WITH PNIC BSA 4)
Absolutely nothing went correct on this one. This is probably because the sample were sitting in the refrigerator for a week exactly. That may have interfered with the complexion of the protein.
WEEK 6 (VIRTUAL SCREENING)
Done with both runs 1 and 2 but didn't have time to analyze them. Well ran run 2 on Thursday but had to rerun it on Friday as there was an error in the run. Also did quite a lot of research on my drug. Re did pnic-bsa4 PCR. Samples are in the fridge i jst have to make a gel and analyze them next week.
Mohammed - put your results in Reverse Chronological order. -- Dr. B
Mohammed's Research
WEEK 13
WEEK 12
Serine Threonine Phosphotase 2C from
http://web.expasy.org/cgi-bin/protparam/protparam
Number of amino acids: 324
Molecular weight: 35931.7
Theoretical pI: 5.21
WEEK 11
This week I finished my PCR squared and completed my PCR cleanup
And I finished up my protein expression.
The Pellet Weight was 2.07g for sample 1 and 2.54 for sample 2.
It took an hour and thirty minutes to get to get to 0.1 absorption
Sample 1 Sample 2
3:00
0.163 0.108
0.193 0.199
0.29 0.28
I forgot to take a picture of the pellet but I completed the procedure and the supernatant is in the 4 degree fridge. Also I had two samples but ended up adding too much lysate to sample one so I did my protein purification with sample 2.
WEEK 10
I was not in lab at all this week, well maybe for like 2 to 3 hours, but i will make the hours up for next week. Had three tests and I got pretty sick so sorry about that.
I was supposed to do PCR squared but I will be finishing that next week.
I did my slides for my presentation and talked to Dr.B about them.
Finally fixed my virtual screening.
I started on my Protein Expression but I left too long of a gap so I need to redo it for next week.
So for next week I will finish Virtual Screening of HF9 Library and most probably one other library now that I can do it myself.
Present
Finish all of the PCR
Finish Protein Expression
Will definitely start cloning but don't know if I will be able to finish.
WEEK 9
Mohammed - I think this should be Week 9 - spit up your Week 8 and 9 results.
Good job on the cloning! - Dr. B
Lane 4: 100bp ladder
Lane 5: 2nd PCR
Lane 6: 1st PCR
Lane 7: 1000kb ladder
Lane 8: 1st PCR
Lane 1: 100 bp ladder
Lane 2: 1000 kb ladder
Lane 3: Primary PCR
Lane 4: Secondary PCR
Lane 1: 100bp ladder
Lane 2: 1kb ladder
Lane 3: 1st PCR
Lane 4: 2nd PCR
WEEK 8
Also ran the first GOLD job. Here is the gold.conf file for the first run.
GOLD CONFIGURATION FILE
AUTOMATIC SETTINGS
autoscale = 0.1
POPULATION
popsiz = auto
select_pressure = auto
n_islands = auto
maxops = auto
niche_siz = auto
GENETIC OPERATORS
pt_crosswt = auto
allele_mutatewt = auto
migratewt = auto
FLOOD FILL
radius = 10
origin = 30.877 16.634 59.271
do_cavity = 1
floodfill_atom_no = 2454
cavity_file = ID_protein.mol2
floodfill_center = atom
DATA FILES
ligand_data_file /home/chem204/DatabasesVDS/HF9PlatesPlates5_9.sdf 10
param_file = DEFAULT
set_ligand_atom_types = 1
set_protein_atom_types = 0
directory = .
tordist_file = DEFAULT
make_subdirs = 0
save_lone_pairs = 1
fit_points_file = fit_pts.mol2
read_fitpts = 0
bestranking_list_filename = BestPP2CvsHF9Run1.1st
FLAGS
internal_ligand_h_bonds = 0
flip_free_corners = 0
match_ring_templates = 0
flip_amide_bonds = 0
flip_planar_n = 1 flip_ring_NRR flip_ring_NHR
flip_pyramidal_n = 0
rotate_carboxylic_oh = flip
use_tordist = 1
postprocess_bonds = 1
rotatable_bond_override_file = DEFAULT
solvate_all = 1
TERMINATION
early_termination = 1
n_top_solutions = 3
rms_tolerance = 1.5
CONSTRAINTS
force_constraints = 0
COVALENT BONDING
covalent = 0
SAVE OPTIONS
save_score_in_file = 1
save_protein_torsions = 1
concatenated_output = PP2CvsHF9Run1.sdf
clean_up_option delete_all_solutions
clean_up_option save_top_n_solutions 1
clean_up_option save_best_ligands 10
clean_up_option delete_redundant_log_files
clean_up_option delete_all_initialised_ligands
clean_up_option delete_empty_directories
clean_up_option delete_rank_file
clean_up_option delete_all_log_files
output_file_format = MACCS
FITNESS FUNCTION SETTINGS
initial_virtual_pt_match_max = 3
relative_ligand_energy = 1
start_vdw_linear_cutoff = 6
score_param_file = DEFAULT
PROTEIN DATA
protein_datafile = /home/chem204/2011/ma32259/Serine Threonine Phosphotase 2C/ID_protein.mol2
Also ran the second GOLD Job
Here is the gold.conf file for the second fun
GOLD CONFIGURATION FILE
AUTOMATIC SETTINGS
autoscale = 1
POPULATION
popsiz = auto
select_pressure = auto
n_islands = auto
maxops = auto
niche_siz = auto
GENETIC OPERATORS
pt_crosswt = auto
allele_mutatewt = auto
migratewt = auto
FLOOD FILL
radius = 10
origin = 30.877 16.634 59.271
do_cavity = 1
floodfill_atom_no = 2454
cavity_file = ID_protein.mol2
floodfill_center = atom
DATA FILES
ligand_data_file /home/chem204/2011/ma32259/Serine Threonine Phosphotase 2C/SDFCOMBINED 10
param_file = DEFAULT
set_ligand_atom_types = 1
set_protein_atom_types = 0
directory = .
tordist_file = DEFAULT
make_subdirs = 0
save_lone_pairs = 1
fit_points_file = fit_pts.mol2
read_fitpts = 0
bestranking_list_filename = BestPP2CvsHF9Run1.1st
FLAGS
internal_ligand_h_bonds = 0
flip_free_corners = 0
match_ring_templates = 0
flip_amide_bonds = 0
flip_planar_n = 1 flip_ring_NRR flip_ring_NHR
flip_pyramidal_n = 0
rotate_carboxylic_oh = flip
use_tordist = 1
postprocess_bonds = 1
rotatable_bond_override_file = DEFAULT
solvate_all = 1
TERMINATION
early_termination = 1
n_top_solutions = 3
rms_tolerance = 1.5
CONSTRAINTS
force_constraints = 0
COVALENT BONDING
covalent = 0
SAVE OPTIONS
save_score_in_file = 1
save_protein_torsions = 1
concatenated_output = PP2CvsHF9Run2.sdf
clean_up_option delete_all_solutions
clean_up_option save_top_n_solutions 1
clean_up_option save_best_ligands 1
clean_up_option delete_redundant_log_files
clean_up_option delete_all_initialised_ligands
clean_up_option delete_empty_directories
clean_up_option delete_rank_file
clean_up_option delete_all_log_files
output_file_format = MACCS
FITNESS FUNCTION SETTINGS
initial_virtual_pt_match_max = 3
relative_ligand_energy = 1
start_vdw_linear_cutoff = 6
score_param_file = DEFAULT
PROTEIN DATA
protein_datafile = /home/chem204/2011/ma32259/Serine Threonine Phosphotase 2C/ID_protein.mol2
Best Ranking for 1st GOLD RUN
Best Ranking List for GOLD RUN 2
WEEK 7
Did the Primer Overlap Assembly as I designed a set of forward and reverse primers for PCR for serine threonine phosphotase 2C
This is the DNA sequence for the particular target.
This is the oligonucleotides(primers) that I obtained
The protein is serine throenine phosphotase 2C and the strain was made for E.Coli 21
For the particular target there are 24 primers
Also in wet lab I completed the primary and secondary PCR and will do PCR squared this coming week.
WEEK 2
WEEK 2 (PCR WITH pGBR22)
Lane 1: skip
Lane 2: 100 bp ladder
Lane 3: PCR of pGBR22 Tube A
Lane 4: PCR of pGBR22 Tube B
Lane 5: PCR of pGBR22 Tube C
Lane 6: PCR of pGBR22 Tube D
WEEK 2 (ANALYZING DNA SEQUENCES)
HUMAN SEQUENCING
NUCLEOTIDE SEQUENCING
COMPARISON OF PURPLE PROTEIN TO pGBR22
a
WEEK 2 RE DIGEST
Lane 2 1kB ladder
Lane 3 uncut pgbr22 plasmid
Lane 4 eco R1
Lane 5 PvuII
Lane 6 EcoR1 + PvuII
WEEK 3 SUBMITTING DNA SEQUENCE
The Requests are entered, your Order Number is: 72169
ICMB Core Facilities
DNA Sequencing
2500 Speedway
MBB 1.426L, A4800
Austin, TX 78712
512 475-7844 begin_of_the_skype_highlighting 512 475-7844 end_of_the_skype_highlighting p
512 471-2149 fax
|| ||
WEEK 3 (PCR WITH pGBR22 AGAIN)
_
Lane 2 1kB ladder
Lane 3 sample a
Lane 4 sample b
Lane 5 sample c
Lane 6 sample d
WEEK 3 (PCR WITH PNIC BSA4)
Lane 2: Ladder 100bp
Lane 3: 1 in 1000 concentration
Lane 4: 1 in 1000 concentration
Lane 5: 1 in 1000 concentration
WEEK 4 (PCR WITH PNIC BSA4)
_
Lane 1: 100bp ladder
Lane 2: 1 in 100 concentration
Lane 3: 1 in 1000 concentration
Lane 4: 1 in 10000 concentration
ALSO IN WEEK 4 COMPLETED THE PYMOL REFRESHER WHICH IS IN GOOGLE DOCS
WEEK 5 (PCR WITH PNIC BSA 4)
WEEK 6 (VIRTUAL SCREENING)
Done with both runs 1 and 2 but didn't have time to analyze them. Well ran run 2 on Thursday but had to rerun it on Friday as there was an error in the run. Also did quite a lot of research on my drug. Re did pnic-bsa4 PCR. Samples are in the fridge i jst have to make a gel and analyze them next week.
Mohammed - put your results in Reverse Chronological order. -- Dr. B